CN111165352A - Novel space breeding tissue culture seedling raising method for gentiana rigescens - Google Patents
Novel space breeding tissue culture seedling raising method for gentiana rigescens Download PDFInfo
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- CN111165352A CN111165352A CN202010034476.2A CN202010034476A CN111165352A CN 111165352 A CN111165352 A CN 111165352A CN 202010034476 A CN202010034476 A CN 202010034476A CN 111165352 A CN111165352 A CN 111165352A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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Abstract
The invention provides a novel method for tissue culture and seedling of gentiana rigescens based on the characteristics of low germination rate and low cutting propagation rate of gentiana rigescens space breeding seeds, which mainly comprises the steps of explant selection and disinfection, cluster bud induction, cluster bud proliferation and rooting seedling culture. The method is simple and easy to implement, has the advantages of short breeding period, high breeding rate, capability of effectively stabilizing the characters of the mother plant and the like, and is the most effective way for producing the space species with the stable characters of the gentiana rigescens in a large scale.
Description
Technical Field
The invention relates to the field of biotechnology of flowers and traditional Chinese medicinal materials, in particular to a tissue culture method for space breeding of gentiana rigescens.
Technical Field
Gentiana rigescens (Gentianararigescens Franch.) belongs to Gentiana of Gentianaceae, is one of the plant sources of Gentiana scabra Bunge recorded in pharmacopoeia of the people's republic of China, and is also the most main source of Gentiana scabra Bunge in the southwest region. The gentiana rigescens is used as a medicine mainly from Yunnan and Sichuan provinces, has the effects of clearing heat and drying dampness and purging liver and gallbladder fire, and is mainly used for treating damp-heat jaundice, vulva swelling and pruritus vulvae, leukorrhagia, eczema and pruritus, liver fire and conjunctival congestion, tinnitus and deafness, hypochondriac pain and bitter taste in mouth, strengthening middle-jiao, convulsion and convulsion. Modern researches believe that the main active ingredient of the medicine is gentiopicroside which has the effects of resisting inflammation, easing pain, resisting anoxia, resisting fatigue and the like.
The gentiana rigescens seeds in 6 months in 2013 enter the space along with Shenshi, more than 200 female parents with different characters are obtained after non-directional variation, and more than 40 female parents with high-quality characters are obtained after screening of flower color, plant type, medicinal material yield and effective component content, and can be used for production of high-quality medicinal materials.
Although the yield of the gentiana rigescens seeds is high, the gentiana rigescens seeds are small (the thousand seed weight is only about 20 mg), the maturity of the seeds is very low, the germination rate of the seeds is extremely low, the seed setting rate and the germination rate of the gentiana rigescens space breeding seeds are lower, the gentiana rigescens space breeding seeds cannot be effectively stored and propagated, on the other hand, as the gentiana rigescens belongs to heterogamy, the filial generation and the maternal characters are greatly different through heterogamy hybridization, stable characters cannot be formed, the gentiana rigescens seed propagation cannot become the best mode of gentiana rigescens space seed propagation, and the cutting propagation and the division propagation have the advantages of extremely low propagation rate and long propagation period. Therefore, the Gentiana rigescens space seeds with stable characters and sufficient quantity can not be obtained in a short time by utilizing the traditional technology. The gentiana rigescens space breeding seedling culture is carried out by adopting the tissue culture breeding technology, so that sufficient seedlings can be obtained in a short period, and the stability of female parent characters can be ensured to the greatest extent.
Disclosure of Invention
Aiming at the defects of the traditional gentiana rigescens space breeding propagation method, the invention provides a tissue culture technology which has simple components, low cost, simple and convenient operation and short production period and can directly produce different gentiana rigescens space breeding varieties in batches.
The invention is realized by the following scheme:
(1) selection of explants: selecting healthy branches without diseases and insect pests, and picking tender branches within 20cm of the ends of the branches as explants;
(2) cleaning explants: removing leaves of the explant obtained in the step (1), cutting the explant into 1-2 cm stem sections with buds, placing the stem sections in saturated soap water for rinsing for 10min, then washing the stem sections with tap water for 3-4 times, and finally washing the stem sections with running water for 1 h;
(3) and (3) disinfection of explants: adding 75% alcohol into the explant obtained in the step (2) in a super clean workbench, shaking for 15-20 s, rinsing with sterile water for 3-4 times, adding a saturated bleaching powder solution, shaking for 28-32 min, rinsing with sterile water for 3-4 times, adding a 0.05% mercuric chloride solution, shaking for 9-11 min, rinsing with sterile water for 6-7 times, and culturing the explant for 7 days, wherein the pollution rate is 17%;
(4) and (3) induction culture: removing two wounds from the explant obtained in the step (3), inoculating the explant into an induction culture medium of MS + KT 5.0-10.0 mg/L + TDZ 0.05-0.15 mg/L + NAA 0.01-0.05 mg/L, and culturing for 40 days in an environment with the temperature of 25 ℃, the light intensity of 2000lx and the light dark 12:12h alternately, wherein the induction rate of the cluster buds is 68%;
(5) and (3) proliferation culture: inoculating the induced bud obtained in the step (4) into an enrichment medium of 5.0-10.0 mg/L MS + KT + 0.01-0.05 mg/L ZT, and culturing for 25 days in an environment with the temperature of 25 ℃, the light intensity of 2000lx and the light dark of 12:12h alternately, wherein the enrichment coefficient is 6.0;
(6) rooting culture: and (3) inoculating the induced bud or the proliferated bud obtained in the step (4) or the step (5) into a rooting culture medium of 1/2MS + NAA 0.5-1 mg/L + IAA 0.5-1.0 mg/L + IBA 1.0-4.0 mg/L + AC1.0g/L, and culturing for 25 days in an environment with the temperature of 25 ℃, the light intensity of 3000lx and the light darkness of 12:12h alternately to obtain a rooting tissue culture seedling, wherein the rooting rate is 94%.
The invention has the beneficial effects that
(1) The invention adopts the tissue culture rapid propagation technology, has simple components, simple and convenient operation and low cost, is carried out under the indoor temperature control and light control nutrition condition, and is not limited by regions and seasons for breeding and seedling;
(2) according to the tissue culture rapid propagation technology adopted by the invention, the initial pollution rate is about 17%, the initial pollution rate is 10-20% lower than that of other gentiana rigescens tissue culture technologies, the initial induction rate in 45 days is about 68%, the initial induction rate is improved by 5-20% compared with that of other gentiana rigescens tissue culture technologies, the 25-day subculture multiplication coefficient is 6.0, the multiplication coefficient is 0.5-1.5 higher than that of other gentiana rigescens tissue culture technologies, the 25-day root induction rate is 94%, the tissue culture technology is similar to that of other gentiana rigescens, but the seedlings are more robust, and the domestication and transplantation survival rate is higher;
(3) the invention aims at space seeds of gentiana rigescens, is a plurality of new seeds obtained by space mutagenesis after carrying god decimal places, has great difference with the physiological characteristics of the traditional gentiana rigescens, and particularly shows that the seed setting rate and the germination rate of the seeds of the gentiana rigescens are far lower than those of the traditional gentiana rigescens, but the application of the existing tissue culture technology in the space seeds of the gentiana rigescens is not as good as that of the invention.
Detailed Description
The present invention will be further described with reference to examples.
Example 1: in a Gentiana rigescens space breeding and breeding base, a breed MB12 bred by Gentiana rigescens space breeding is sprayed with 1000-fold liquid of 50 percent carbendazim to healthy plant without diseases and insect pests in advance and is bagged to reduce the quantity of carried bacteria, when explants are collected, the explant is soaked in 75 percent alcohol for more than 10s, and branches within 10cm of the end part are cut;
removing leaves from the branches and shearing the branches into stem sections with buds of about 1-2 cm in a laboratory, rinsing the stem sections with the buds in saturated soap water for 10min, washing the stem sections with tap water for 3-4 times until no foam exists on the surface, and then flushing the stem sections with small running water for 1 hour;
transferring the cleaned stem segments into a sterile bottle in a clean bench, adding 75% alcohol, shaking for 20s, rinsing with sterile water for 3-4 times, adding a saturated bleaching powder solution, shaking for 30min, rinsing with sterile water for 3-4 times, transferring to another sterile bottle, adding a 0.05% mercuric chloride solution, shaking for 10min, washing with sterile water for 7 times, and carrying out explant culture for 7 days, wherein the pollution rate is 18.2%;
obliquely inserting the sterilized explant into an induction culture medium MS + KT5.0 mg/L + TDZ 0.05mg/L + NAA0.01mg/L according to physiological polarity, and culturing for 45 days at the temperature of 25 ℃, under the light intensity of 2000lx and in the dark for 12:12h to obtain a robust induction bud with the induction rate of 64.6%;
cutting the induced cluster buds into stem sections with 1-2 buds, obliquely inserting the stem sections into a multiplication medium MS + KT 10.0mg/L + ZT0.01mg/L according to polarity, and culturing for 25 days in an environment with the temperature of 25 ℃, the light intensity of 2000lx and the light dark of 12:12h alternately to obtain a large amount of robust cluster buds, wherein the multiplication coefficient is 5.91;
cutting the proliferated buds into stem segments with 2-3 buds, culturing in a rooting culture medium with the polarity difference of 1/2MS + NAA1.0mg/L + IAA 1.0mg/L + IBA 4.0mg/L + AC1.0g/L for 25 days at 25 ℃, under the condition of 3000lx light intensity and 12:12h light intensity alternately to obtain rooted seedlings, wherein the roots generally comprise 2-3 roots, the roots are thick and strong, and the rooting rate is 93.3%.
Example 2:
spraying a healthy plant without diseases and insect pests with 1000-fold liquid of 50% carbendazim for one month in advance on a variety Z-1-2 bred by Gentiana rigescens space breeding base, bagging to reduce the amount of carried bacteria, soaking the collected explant in 75% alcohol for more than 10s, and shearing branches with the end part within 20 cm;
removing leaves from the branches and shearing the branches into stem sections with buds of about 1-2 cm in a laboratory, rinsing the stem sections with the buds in saturated soap water for 10min, washing the stem sections with tap water for 3-4 times until no foam exists on the surface, and then flushing the stem sections with small running water for 1 hour;
transferring the cleaned stem segments into a sterile bottle in a clean bench, adding 75% alcohol, shaking for 15s, rinsing with sterile water for 3-4 times, adding a saturated bleaching powder solution, shaking for 32min, rinsing with sterile water for 3-4 times, transferring to another sterile bottle, adding a 0.05% mercuric chloride solution, shaking for 11min, washing with sterile water for 7 times, and carrying out explant culture for 7 days, wherein the pollution rate is 16.5%;
obliquely inserting the sterilized explants into an induction culture medium MS + KT 8.0mg/L + TDZ 0.10mg/L + NAA0.05mg/L according to physiological polarity, and culturing for 45 days at the temperature of 25 ℃, under the light intensity of 2000lx and in the light dark for 12:12h alternately to obtain robust induced buds, wherein the induction rate is 71.2%;
cutting the induced cluster buds into stem sections with 1-2 buds, obliquely inserting the stem sections into a multiplication medium MS + KT 10.0mg/L + ZT0.05mg/L according to polarity, and culturing for 25 days in an environment with the temperature of 25 ℃, the light intensity of 2000lx and the light dark of 12:12h alternately to obtain a large number of strong multiplication buds, wherein the multiplication coefficient is 6.2;
cutting the proliferated buds into stem segments with 2-3 buds, culturing in a rooting culture medium 1/2MS + NAA0.5 mg/L + IAA0.5mg/L + IBA 2.0mg/L + AC1.0g/L for 25 days at 25 ℃, under 3000lx light intensity and 12:12h light intensity alternately to obtain rooted seedlings, wherein the roots are 2-4 in most, the roots are thick and strong, and the rooting rate is 95.4%.
Example 3: in a Gentiana rigescens space breeding base, spraying a healthy plant without diseases and insect pests with 1000-fold liquid of 50% carbendazim in advance on a variety MB24 bred by Gentiana rigescens space breeding, bagging to reduce the amount of carried bacteria, soaking the collected explant in 75% alcohol for more than 10s, and cutting branches with the end part within 20 cm;
removing leaves from the branches and shearing the branches into stem sections with buds of about 1-2 cm in a laboratory, rinsing the stem sections with the buds in saturated soap water for 10min, washing the stem sections with tap water for 3-4 times until no foam exists on the surface, and then flushing the stem sections with small running water for 1 hour;
transferring the cleaned stem segments into a sterile bottle in a clean bench, adding 75% alcohol, shaking for 20s, rinsing with sterile water for 3-4 times, adding a saturated bleaching powder solution, shaking for 30min, rinsing with sterile water for 3-4 times, transferring to another sterile bottle, adding a 0.05% mercuric chloride solution, shaking for 10min, washing with sterile water for 7 times, and carrying out explant culture for 7 days, wherein the pollution rate is 16.0%;
obliquely inserting the sterilized explant into an induction culture medium MS + KT5.0 mg/L + TDZ 0.05mg/L + NAA0.01mg/L according to physiological polarity, and culturing for 45 days at the temperature of 25 ℃, under the light intensity of 2000lx and in the light dark for 12:12h alternately to obtain a robust induction bud with the induction rate of 71.2%;
cutting the induced cluster buds into stem sections with 1-2 buds, obliquely inserting the stem sections into a multiplication medium MS + KT 10.0mg/L + ZT0.01mg/L according to polarity, and culturing for 25 days in an environment with the temperature of 25 ℃, the light intensity of 2000lx and the light dark of 12:12h alternately to obtain a large amount of robust cluster buds, wherein the multiplication coefficient is 6.8;
cutting the proliferated buds into stem segments with 2-3 buds, culturing in a rooting culture medium 1/2MS + NAA1.0mg/L + IAA 1.0mg/L + IBA 4.0mg/L + AC1.0g/L for 25 days at 25 ℃ under 3000lx light intensity and 12:12h light intensity alternately to obtain rooted seedlings with 2-4 roots, thick roots and 94.8% rooting rate.
Claims (5)
1. The novel method for space breeding, tissue culture and seedling raising of gentiana rigescens is characterized by comprising the following steps:
(1) selection and cleaning and disinfection of explants: taking healthy stems of Gentiana rigescens Taikong plant ends within 20cm, cutting into 1cm stem sections with buds after removing leaves, washing with soap water for 10min, washing with tap water for more than 1h, and sterilizing with 75% alcohol, saturated bleaching powder solution and 0.05% mercuric chloride solution in sequence;
(2) inducing cluster buds: inoculating the explant obtained in the step (1) into an MS culture medium containing KT, TDZ and NAA, and culturing for 40 days at the temperature of 25 ℃ under the condition of 2000lx light intensity and 12:12h light intensity alternately to induce cluster buds;
(3) and (3) multiplication of cluster buds: inoculating the induced bud obtained in the step (2) into an MS culture medium containing KT and ZT, and culturing for 45 days in an environment with the temperature of 25 ℃, the light intensity of 2000lx and the light dark of 12:12h alternately to obtain a proliferation bud;
(4) rooting of the proliferated buds: and (3) inoculating the induced bud or the proliferation bud obtained in the step (1) or the step (2) into a 1/2MS culture medium containing NAA, IAA and IBA, and culturing for 35 days in an environment with the temperature of 25 ℃, the light intensity of 3000lx and the light dark of 12:12h alternately to obtain the rooting tissue culture seedling.
2. The space breeding tissue culture seedling raising new method of gentiana rigescens according to claim 1, characterized in that 75% alcohol is shaken for 15-20 s, saturated bleaching powder solution is shaken for 28-32 min, and 0.05% mercuric chloride solution is shaken for 9-11 min.
3. The novel method for space breeding, tissue culture and seedling raising of gentiana rigescens according to claim 1, wherein the cluster bud induction medium in the step (2) is MS + KT 5.0-10.0 mg/L + TDZ 0.05-0.15 mg/L + NAA 0.01-0.05 mg/L.
4. The novel method for space breeding, tissue culture and seedling raising of gentiana rigescens according to claim 1, wherein the multiplication medium in the step (3) is MS + KT 5.0-10.0 mg/L + ZT 0.01-0.05 mg/L.
5. The space breeding tissue culture seedling raising new method of gentiana rigescens according to claim 1, characterized in that the rooting medium in step (4) is 1/2MS + NAA 0.5-1 mg/L + IAA 0.5-1.0 mg/L + IBA 1.0-4.0 mg/L + AC1.0 g/L.
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Cited By (4)
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| CN111616048A (en) * | 2020-04-27 | 2020-09-04 | 云南省农业科学院药用植物研究所 | Novel tissue culture and rapid propagation method for asparagus cochinchinensis |
| CN112293256A (en) * | 2020-11-11 | 2021-02-02 | 中国长江三峡集团有限公司 | Space China rose tissue culture propagation method |
| CN115606504A (en) * | 2022-12-19 | 2023-01-17 | 云南省农业科学院药用植物研究所 | Cultivation method of gentiana rigescens polyploidy |
| CN115612708A (en) * | 2022-12-19 | 2023-01-17 | 云南省农业科学院药用植物研究所 | Method for producing gentiopicroside by using callus of gentiana rigescens |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111616048A (en) * | 2020-04-27 | 2020-09-04 | 云南省农业科学院药用植物研究所 | Novel tissue culture and rapid propagation method for asparagus cochinchinensis |
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| CN115606504A (en) * | 2022-12-19 | 2023-01-17 | 云南省农业科学院药用植物研究所 | Cultivation method of gentiana rigescens polyploidy |
| CN115612708A (en) * | 2022-12-19 | 2023-01-17 | 云南省农业科学院药用植物研究所 | Method for producing gentiopicroside by using callus of gentiana rigescens |
| CN115606504B (en) * | 2022-12-19 | 2023-05-05 | 云南省农业科学院药用植物研究所 | Polyploid cultivation method for gentiana rigescens |
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Application publication date: 20200519 |